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ORIGINAL ARTICLE

Effects of Steroids and Retinoids on Wound Healing


Corinna Wicke, MD; Betty Halliday; Daniel Allen, MD; Nanette S. Roche, MS; Heinz Scheuenstuhl;
Martin M. Spencer, MD, PhD; Anita B. Roberts, PhD; Thomas K. Hunt, MD

Hypothesis: Anti-inflammatory corticosteroids sig-

nificantly impair wound healing. Retinoids partially,


but significantly, reverse this effect. Little is known
about the mechanism of steroid retardation or retinoid
reversal. We hypothesized that corticosteroids lower
transforming growth factor-b (TGF-b) and insulinlike growth factor-I (IGF-I) levels and tissue deposition in wounds and that retinoids stimulate
corticosteroid-impaired TGF-b and IGF-I release and
collagen production.
Design: Randomized controlled trial.
Setting: Wound healing research laboratory.
Participants: Animal study.
Interventions: Four wire mesh wound cylinders
were implanted subcutaneously into the backs of 72
male Sprague-Dawley rats. Wound healing was
impaired by a single subcutaneous injection of 6 mg of
methylprednisolone acetate (Depo-Medrol). Two
preparations of retinoids were used in separate experi-

From the Departments


of Surgery, University of
Tubingen, Tubingen, Germany
(Dr Wicke), University
of California, San Francisco
(Ms Halliday, Mr Scheuenstuhl,
and Dr Hunt); Division
of Plastic Surgery, University
of California, Davis (Dr Allen);
Laboratory of Cell Regulation
and Carcinogenesis National
Cancer Institute, Bethesda, Md
(Drs Roche and Roberts); and
the Laboratory of Growth and
Development, Davies Medical
Center, San Francisco
(Dr Spencer). The authors have
no commerical, proprietary, or
financial interest in the
products and companies
described in this article.

ments: all-trans-retinoic acid and 9-cis-retinoic acid


that were fed orally.
Main Outcome Measures: Hydroxyproline content
was measured in the healing tissue and TGF-b and IGF-I
levels were analyzed in the wound fluid.
Results: Methylprednisolone treatment significantly de-

creased TGF-b and IGF-I levels in the wound fluid and


hydroxyproline content in the tissue (P,.05). Oral alltrans- and 9-cis-retinoic acid partially reversed the TGF-b
and IGF-I decrease and significantly increased hydroxyproline content toward normal levels (P,.05). Oral alltrans-retinoic acid enhanced collagen deposition, TGF-b
and IGF-I levels over normal chow fed control animals
(P,.05).
Conclusions: Steroids and retinoids have antagonistic
effects on growth factors and collagen deposition in wound
healing. These effects can be relevant for treatment
options in a clinical setting.

Arch Surg. 2000;135:1265-1270

NTI-INFLAMMATORY gluco-

corticosteroids markedly
affect most aspects of
wound healing. When administered sufficiently
early after injury, high corticosteroid levels delay the appearance of inflammatory
cells, fibroblasts, the deposition of ground
substance, collagen, regenerating capillaries, contraction, and epithelial migration.1
Retinoids have the unique ability to
reverse these inhibitory effects, except for
wound contraction. Impairment of the inflammatory response, tensile strength, and
collagen accumulation in cutaneous
wounds after steroid treatment are partially, but significantly, reversed by retinoid administration (retinyl ester, retinol, and/or retinoic acid). 2 Similar
observations were made studying flexor
tendon repair,3 healing of rat femoral frac-

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1265

tures,4 vessel repair,5 and healing of intestinal anastomoses.6 Although steroid retardation of healing is a significant clinical
problem and a commonly used laboratory model in the study of repair processes, little is known about the mechanisms of either steroid retardation or
reversal by retinoids.
Retinoids are global regulators of
the growth and differentiation of many
cell types. It is now appreciated that all
actions of retinoids are mediated through
2 families of nuclear receptors belonging
to the steroid hormone receptor superfamily.7,8 These receptors, termed retinoic acid receptors (RARs) and retinoid
X receptors (RXRs), bind and transactivate distinct response elements of
numerous genes required to maintain
differentiation and proliferation of epithelial tissues. The ligand for these
receptors is retinoic acid. Other, more

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MATERIALS AND METHODS


Four wire mesh wound cylinders were implanted under
the dorsal skin of 72 male Sprague-Dawley rats (weighing 350 g). Wire mesh cylinders were chosen to obtain
wound fluid and wound tissue simultaneously and to
assess wound fluid at different times. Wound healing
was impaired by a single subcutaneous injection of 6 mg
of methylprednisolone acetate at the time of surgery.
This time point was chosen since the inhibitory effect of
glucocorticosteroids on wound healing is most pronounced when administration of the hormone begins
just prior to the onset of inflammation in the healing
process.26
The following preparations of retinoids were used:
(1) all-trans-retinoic acid (120 mg of all-trans-retinoic acid
per kilogram of diet); 20 g of diet fed daily (approximately
2.4 mg of all-trans-retinoic acid per rat per day); and
(2) 9-cis-retinoic acid (120 mg of 9-cis-retinoic acid per kilogram of diet); 20 g of diet fed daily (approximately 2.4
mg of 9-cis-retinoic acid per rat per day).
The animals were assigned to 4 treatment arms:
(1) corticosteroids alone, (2) either of the 2 retinoids alone,
(3) corticosteroids and either of the 2 retinoids, and
(4) control animals. The controls were fed regular rat chow
(Purina Ground Laboratory Chow; Purina, Ralston Purina,
Atlanta, Ga, containing 17.6 IUapproximately 5 gof
vitamin A per gram). All animals drank water ad libitum.
The study protocol was approved by the University of California, San Francisco Committee of Animal Research.
Wound fluid was harvested by sterile aspiration with
a syringe at different times in the various experiments. Transforming growth factor b levels were assayed on day 3, 7,
11, 15, and/or 17 using a nonradioactive bioassay based on
its ability to induce expression of plasminogen activator inhibitor-1 in mink lung epithelial cells stably expressing a

reduced metabolites function to control the storage,


trafficking, and conversion to the metabolically
unstable active form, retinoic acid.
Certain actions of the retinoids on cells are now
known to be mediated through regulation of the levels
of expression of secreted growth factors and/or their receptors.9-11 Two of the peptides known to be regulated
by retinoic acid include the transforming growth factor
b (TGF-b) isoforms, TGF-b1, 2, and 3, and insulin-like
growth factor I (IGF-I).7,12 Each of these growth factors
has been shown to regulate important phases of wound
healing on its release from platelet a granules.13
The TGF-bs and IGF-I are synthesized and secreted
by cells constituting the newly formed granulation tissue, they can each act as local mediators of cellular response and they can regulate interactions between cells
based on the ubiquitous nature of their receptors.14 Platelets release 2 distinct forms of latent TGF-b: the small
form containing TGF-b and the latency associated protein is directly released into the supernatant of clotted
blood, whereas the large form containing mature TGF-b,
latency associated protein, and latent TGF-bbinding protein, which binds latency associated protein covalently,
remains associated with the clot and is made available
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1266

portion of the plasminogen activator inhibitor-1 promoter linked to luciferase as described.27


Wound fluid samples were diluted 1:25 in serumfree medium to a final protein concentration of 50 to 100
g/mL and heat activated (80C for 8 minutes). Samples
were then chilled on ice and 200 g of bovine serum albumin was added as carrier. Each sample was assayed individually in a 6-point dilution curve in 24-well plates and
its activity compared with a standard curve of recombinant human TGF-b1 run in the same assay to determine
the concentration of active TGF-b in the wound fluid. Each
sample was assayed in 2 or 3 replicate assays and the results averaged. The wound fluid from each wound cylinder was assessed individually for TGF-b content. Insulinlike growth factor-I levels were measured at day 17 by
radioimmunoassay. Insulin-like growth factor-I was separated from its binding proteins prior to radioimmunoassay by the formic acidacetone extraction method.28 For
assessment of IGF-I levels only, the wound fluid from all
4 wound chambers was pooled for each animal. The animals were killed on day 17 and the cylinders were explanted. All accessible wound tissue was removed mechanically from the implants. Hydroxyproline content of the tissue
within the wound cylinders was assessed by highperformance liquid chromatography with a modified method
after Lindblad and Diegelmann.29
Immunohistochemistry was performed on fullthickness skin sections obtained from the back of the animals from an unwounded area. The sections were stored
in 10% formalin solution and stained with the 3 anti
TGF-b antibodies (antiTGF-b1, antiTGF-b2,
antiTGF-b3). Rabbit polyclonal antibodies to the TGF-bs
were used as follows: TGF-b1, anti-P (1-30) LC at 7 g/
mL, TGF-b2, anti-P (50-75) at 1.5 g/mL, TGF-b3, anti-P
(50-60) at 2.4 g/mL.30 The results were statistically analyzed by SuperANOVA (Department of Biostatistics, University of California, San Francisco).

only slowly as the clot is proteolyzed.15 Transforming


growth factor-b affects all phases of the healing process,
including the inflammatory response, angiogenesis, and
matrix deposition.16 Transforming growth factor-b is
mitogenic for fibroblasts and stimulates the production
of fibronectin and collagen. Its effects on the extracellular matrix are more complex and more profound than
that of any other growth factor. In animals, TGF-b
enhances strength in incisional wounds.17 It also corrects the wound healing defect created by age or corticosteroid administration.18 No mechanism beyond the fact
that inflammation is restored has been offered. Topical
and systemic TGF-b administration improves healing in
dermal as well as nondermal sites such as bone19 and
intestine.20
Insulin-like growth factor I is a major regulator of
growth and development and of wound healing.21 Wounds
deprived of 90% of their IGFs by hypophysectomy show
a marked impairment in cell replication and deposition
of collagen and a decrease of wound macrophages.22,23
Insulin-like growth factor-I has direct actions on fibroblasts, endothelial, and epithelial cells.
The mechanisms whereby the retinoids regulate the
activity of the TGF-bs and IGF-I are myriad and include
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2000 American Medical Association. All rights reserved.

both transcriptional and posttranscriptional mechanisms as well as regulation of the activity of the mature
peptides. For example, it has been demonstrated that retinoids control not only the stability of messenger RNAs
encoding the TGF-b isoforms, but also regulate activation of TGF-b from its latent forms,24 and, in certain instances, regulate the expression of the cell surface receptors for TGF-b.7,9 Similarly, retinoids have been shown
to regulate not only synthesis and secretion of the IGFs,
but, in certain cases, synthesis of IGF-binding protein as
well.25
Based on the known antagonistic effects of steroids
and retinoids on wound healing, and on the welldocumented effects of retinoids in regulating the activity
of peptide growth factors, we proposed that steroid
treatment would lower basal levels of TGF-b and IGF-I
and would suppress tissue deposition in wounds. Furthermore, we proposed that retinoids would antagonize
these suppressive effects by their ability to stimulate
corticosteroid-impaired TGF-b and IGF-I release and
thereby stimulate tissue deposition. To test this hypothesis, we have examined the effects of methylprednisolone acetate Depo-Medrol treatment on the levels of
TGF-b and IGF-I in wound fluid and on tissue deposition in wounds, and have assessed the ability of various
retinoids to antagonize this effect.

IGF-I LEVELS IN WOUND FLUID


Methylprednisolone treatment significantly decreased
IGF-I levels in wound fluid ( Figure 3 ). Oral alltrans-retinoic acid significantly reversed the steroidinduced IGF-I decrease toward control levels (Figure
3A). Oral all-trans-retinoic acid given alone significantly increased IGF-I levels beyond control levels
(Figure 3A). Oral 9-cis-retinoic acid also significantly
reversed the steroid-induced IGF-I decrease (Figure
3B).
COLLAGEN DEPOSITION IN HEALING TISSUE
Methylprednisolone treatment significantly decreased hydroxyproline content in the wound cylinders by approximately 50% compared with controls at day 17 (Figure 4).
Feeding the steroid-treated animals with all-transretinoic acid increased hydroxyproline content toward
normal levels to within approximately 80% of controls
at day 17 (Figure 4A). Oral all-trans-retinoic acid alone
significantly increased hydroxyproline content beyond
normal levels when compared with animals fed the control diet at day 17 (Figure 4A). Feeding the steroidtreated animals with 9-cis-retinoic acid significantly
increased hydroxyproline content toward normal levels

RESULTS

120

TGF-b LEVELS IN WOUND FLUID


TGF-, ng/mL

100

Tranforming growth factor-b levels in wound fluid


were significantly reduced after 7, 11, 15, and 17 days
in rats treated with steroids when compared with controls (Figure 1 and Figure 2). Oral all-trans-retinoic
acid reversed the steroid-suppressed TGF-b levels
toward normal levels to within approximately
80% of control levels (Figure 2A). Oral all-transretinoic acid given alone significantly increased TGF-b
levels beyond normal when compared with controls
(Figure 2A). Oral 9-cis-retinoic acid also reversed
steroid-suppressed TGF-b levels toward normal to
within approximately 80% of control levels (Figure
2B).
250

80
60

11

15

0
3

Time, d

Figure 1. Transforming growth factor-b (TGF-b) levels in wound fluid over


the course of 15 days. Asterisks indicate P,.05, n=48 cylinders per group.
125

40
20

100

-R
Ac etin
id oic
Di
et

9-

9Ac -R
id eti
/S no
te ic
ro
id

nt
ro

St

l-

cis

cis

Al

Al

l-

er
St

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Al

on

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tra

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id eti
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25

50

50

on

100

75

Al

oi
ds

150

er

TGF-, ng/mL

200

TGF-, ng/mL

Steroid-Treated Rats
Control

Rat Group

Rat Group

Figure 2. Transforming growth factor-b (TGF-b) levels in wound fluid at day 17 in all-trans-isomer (A) and 9-cis-isomer (B). Asterisks indicate P,.05, n=48
cylinders per group. See Materials and Methods section for an explanation of the 4 treatment arms of this study.

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150

150

100

-R
Ac etin
id oic
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A

cis

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9-

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id eti
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t e ic
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id

50

lo
ne

50

9Ac -R
id eti
/S no
t e ic
ro
id

lo
ne

IGF-I, ng/mL

iet

IGF-I, ng/mL

100

Rat Group

Rat Group

Figure 3. Insulin-like growth factor-I (IGF-I) levels in wound fluid at day 17 in all-trans-isomer (A) and 9-cis-isomer (B). Asterisks indicates P,.05, n=9 animals
per group. See Materials and Methods section for an explanation of the 4 treatment arms of this study I.

1500

800

1250

600

HOPro, g

HOPro, g

1000
750

400

500
200
250

l-

e
Ac tin
id oiic
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-R

cis

9-

Al
ds
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Rat Group

Rat Group

Figure 4. Hydroxyproline (HOPro) content in the wound cylinders at day 17 in all-trans-isomer (A) and 9-cis-isomer (B). Asterisks indicate P,.05, n=48 cylinders
per group. See Materials and Methods section for an explanation of the 4 treatment arms of this study.

to within approximately 80% of controls at day 17 (Figure 4B).

treated group (Figure 5). As previously shown, systemic retinoic acid alone induced expression of all TGF-b
isoforms in the epidermis in these experiments.10

IMMUNOHISTOCHEMISTRY
The histological features and immunohistochemical staining patterns reflected the biochemical results and showed
the systemic effects of steroid and/or retinoid treatment
on TGF-b expression. At day 17 control sections of the
skin stained with the TGF-b1 antibody showed maximal staining of hair follicles and muscles. Control sections of the skin stained with the TGF-b2 antibody showed
similarly distributed staining of epidermis, dermis, and
muscle. The muscle staining was less pronounced than
in the control sections stained with TGF-b1 antibody.
Control sections of the skin stained with the TGF-b3 antibody showed the maximum intensity of staining in the
epidermis and a granular distribution of the growth factor. Skin sections from animals treated with steroids
showed thinning of the epidermal layer, fewer hair follicles, and less staining of all 3 TGF-b isoforms. Sections of dermal wounds from animals that received supplemental retinoids showed thicker epidermis, more hair
follicles per microscopic field, and intensified staining
for all 3 TGF-b isoforms when compared with the steroid(REPRINTED) ARCH SURG/ VOL 135, NOV 2000
1268

COMMENT

Corticosteroid hormones are widely used clinically to treat


a variety of diseases by suppressing inflammation and immune functions. Soon after the discovery of the therapeutic potential of corticosteroids, their adverse effects
on wound healing became evident.1 Subsequently, impaired healing during glucocorticoid therapy has become a serious clinical problem. This is well recognized
and steroid-retarded repair has been used frequently as
a test for the effect of various agents including TGF-b
and IGF-I.18
The depressive effect of steroids on wound healing
has long been presumed to depend on the postponement of the inflammatory reaction, without which the
healing sequence cannot proceed. Ehrlich et al2 serendipitously noted that glucocorticoid-mediated reductions in inflammatory cell infiltration, fibroplasia, and
deposition of collagen fibers are prevented by concurrent vitamin A therapy. The proposed mechanism is an
antagonistic effect of corticosteroids and vitamin A on
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2000 American Medical Association. All rights reserved.

multiple inflammatory components. However, no other


mechanistic detail has been added since then.
Glucocorticosteroids decrease collagen synthesis also
in unwounded connective tissues and fibroblast cell culture. The decrease of type I collagen synthesis caused by
steroids has been attributed to a decrease of the steadystate level of total cellular type I procollagen messenger
RNAs. Both glucocorticoids and retinoids regulate a2type I procollagen promoter activity.31
Retinoids regulate the expression of secreted growth
factors. They increase the levels of secreted TGF-b isoforms by as much as 50-fold in certain cells such as keratinocytes.7 Nuclear run-on experiments have confirmed that this effect is not transcriptional, a result
consistent with the lack of identifiable retinoid response elements in the promotors for the TGF-b isoforms. Rather, the increased expression seems to derive
from an increased half-life of the TGF-b messenger RNAs
and from increased translational efficiency.32 Treatment
of cells with retinoids also increases the proportion of
TGF-b that is secreted in the active form. At least for certain cells this can be correlated with the ability of retinoids to increase the expression of transglutaminase, one
of the components in the activation mechanism.33
The goal of this study was to identify whether the above
data could be used to explain the effects of corticosteroids
and retinoids during wound healing. The results show the
predicted suppressive effects of steroids and the stimulatory effects of retinoids on the expression of TGF-b and
IGF-I. Suppression and/or stimulation of TGF-b and IGF-I
expression is paralled by a decrease and/or an increase in
collagen deposition.
All-trans-retinoic acid significantly increased hydroxyproline levels beyond control levels, an effect that
could not be shown with the 9-cis-isomer. This information might be valuable for further studies and could
be significant for therapy of healing deficits. It is tempting to speculate that all-trans-retinoic acid might actually enhance normal healing.
Differences in the pharmacokinetic properties of
orally administered all-trans-retinoic acid and 9-cisretinoic acid have been described.34 In rat breast cancer
prevention the 9-cis-isomer is clearly superior to the alltrans-isomer.35
The 9-cis-isomer has high affinity for both the RAR
and RXR families of retinoid receptors, whereas alltrans-RA binds only to the RAR receptors. Potentially,
broader receptor activation may contribute to the greater
activity of the 9-cis-isomer. Additionally, 9-cis-retinoic
acid does not induce its own oxidative inactivation to the
same extent as all-trans-retinoic acid. Further studies on
the comparative pharmacokinetics of 9-cis-retinoic acid
and all-trans-retinoic acid as well as their activation of
target genes might be useful for enhancing the control
that surgeons have over wound healing.
We attempted but could not repeat prior data showing that parenteral vitamin A palmitate has the same effects as seen for orally fed all-trans- and 9-cis-retinoic acid.2
Parenteral (subcutaneous) vitamin A did not increase the
suppressed TGF-b or IGF-I levels in the wound fluid over
17 days (data not shown). In higher concentrations parenteral vitamin A led to toxic effects including weight
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1269

Figure 5. A, Skin section from control animal, transforming growth


factor-b3 (TGF-b3) immunohistochemistry, 3100 magnification. B, Skin
section from steroid-treated animal, TGF-b3 immunohistochemistry, 3100
magnification. C, Skin section from steroid/9-cis retinoic acidtreated
animal, TGF-b3 immunohistochemistry, 3100 magnification. D, Skin section
from 9-cis retinoic acidtreated animal, TGF-b3 immunohistochemistry,
3100 magnification.

loss. According to the manufacturer, the parenteral retinoid preparations used in earlier experiments, which are
now unavailable, were different mixtures of the various
retinoid metabolites and isomers. The palmitate ester is
metabolized differently than other forms, and this difference rather than the route of administration, in our
opinion, best explains the failure of the parenteral preparation to share the vulnerary results of the orally given
forms.
The immunohistochemical studies were performed for 2 reasons. First, it was necessary to demonstrate that the animals had developed significant systemic effects of the steroids despite the short treatment
duration. Second, we wished to test a long-held hypothesis that systemic retinoids given in treatment of steroidsuppressed repair might diminish the therapeutic potential for which the steroids are being given. Clearly, the
animals were significantly affected, and there is potential for diminishing the desired effects of steroid therapy.
These observations raise several caveats. First, treatment of wound failure in this circumstance should be cautious when interruption of steroid effect might become
problematic. Local retinoid therapy to wounds is also clinically effective. Second, systemic use should be shortWWW.ARCHSURG.COM

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term recognizing that excess vitamin A is stored in the


liver. It is unknown whether systemic retinoids might be
used to forestall wound failure when interruption of the
steroid effect is not critical as for patients with lowgrade rheumatoid arthritis who are being prepared for
operations. However, these data also raise the idea that
retinoids might be used to ameliorate signs and symptoms of Cushings disease or even hasten recovery from
Cushings syndrome after the need for steroid therapy is
ended.
CONCLUSIONS

The results of this study indicate that steroids reduce


TGF-b and IGF-I production in wounds and that collagen deposition suffers by that mechanism. Conversely,
retinoids enhance steroid-retarded healing toward normal levels by restoring TGF-b and IGF-I levels and thereby
reinvigorate collagen production. These effects might well
be shared by other growth factors and substances that
are relevant for the healing process. This study implies
that glucocorticosteroids, retinoids, and the TGF-b family share similar pathways in controlling cell differentiation and proliferation.
Corresponding author: Corinna Wicke, MD, Department of
Surgery, University of Tubingen, Hoppe-Seyler-Strasse 3,
D-72076 Tubingen, Germany (e-mail: Corinna.Wicke@med
.uni-tuebingen.de).
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